PROJECT SUMMARY Melanoma is a highly metastatic cancer of neural crest origin. Despite recent therapeutic advances, Stage IV melanoma has a three-year survival rate of 30-40%. Melanoma Brain Metastasis (MBM), the largest cause of melanoma morbidity and mortality, is clinically evident in approximately half of patients with metastatic disease. This is the highest rate of brain metastasis among cancers, demonstrating melanoma?s unique ability to colonize the brain. MBM patients are less responsive to melanoma therapies and represent an unmet clinical need. Despite several research efforts, the mechanisms that mediate MBM remain poorly understood. We have characterized a novel model system for the study of MBM utilizing three pairs of patient-derived Short Term Cultures (STCs). Each STC pair consists of a brain metastasis (BM) and non-brain metastasis (NBM) derived STC from the same patient, providing an isogenic background for the observation of changes that occurred as a result of the MBM process in patients. We performed unbiased mass spectrometry proteomics analysis of the STC pairs, which revealed a striking alteration in BM vs pair NBM STCs in proteins related to mitochondrial metabolism and Amyloid Precursor Peptide (APP) processing, two interconnected processes that are disrupted in neurodegeneration. Our preliminary in-vitro studies showed increased mitochondrial ATP generation, fusion, and electron density in BM vs pair NBM STCs. We also observed increased APP-specific gamma-secretase activity and amyloid beta secretion in BM vs pair NBM STCs. Through intra-cardiac injection in immunocompromised mice, we showed that silencing of APP in a STC dramatically inhibited formation of brain metastasis without affecting metastasis to other organs, demonstrating that APP plays a critical role in MBM The proposed research plan will define how APP functions in MBM. Proposed in-vivo experiments with analysis of brains at different time points will define which stage of the MBM process APP is important for. Mutant forms of APP with known defects in APP processing will be expressed in an APP knockout STC. By analyzing the ability in-vivo of the various APP mutants to rescue inhibition of MBM by APP loss, we will identify which form of APP functions in MBM. Additionally, the role of APP MBM metabolism and its communications with the brain microenvironment will be assessed through various in-vitro assays. Together, the proposed experiments will characterize the mechanism by which APP functions as a critical mediator of MBM, providing new information about the poorly understood MBM process. The studies will also explore a previously unknown and intriguing mechanistic connection between Alzheimer?s disease and MBM, possibly generating new insights into the pathology of neurodegeneration. Furthermore, this research plan may reveal APP processing as a novel therapeutic target for MBM treatment.